Ralstonia mannitolilytica: an emerging multidrug-resistant opportunistic pathogen in a tertiary care hospital setting

Introduction. Ralstonia mannitolilytica is a rare opportunistic pathogen capable of causing a serious infection in immunocompromised patients. Our objective was to describe all cases of R. mannitolilytica bloodstream infection identified within 2 years at our tertiary care centre, focusing on clinical characteristics, risk factors, antibiotic sensitivity patterns, management and outcomes. Case Series. We compiled a descriptive case series including 14 non-duplicate R. mannitolilytica isolates obtained from bloodstream infection samples from the microbiology laboratory of a tertiary care centre from June 2019 to June 2021. All isolates were initially identified based on their morphological properties and biochemical reactions, and then underwent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) examination for confirmation of identity. Antibiotic susceptibility testing was performed using the Kirby–Bauer disc diffusion method and Vitek 2. All 14 patients presented with symptoms of fever and/or chills, and a positive blood culture for R. mannitolilytica . After 48 h of incubation, no Ralstonia growth was reported from any of the current environmental or pharmaceutical water samples. Chemotherapy (9/14), mechanical ventilation (4/14), steroid use (2/14) and diabetes mellitus (1/14) were associated risk factors in our patients. The antibiotic sensitivity panel showed maximum resistance to aminoglycosides (64.3%) and no resistance to cefoperazone/sulbactum. Patients received treatment with cefoperazone/sulbactum and meropenem or ceftazidime. Thirteen patients recovered with antibiotic therapy and one patient succumbed to his illness. Conclusion. R. mannitolilytica can cause bloodstream infections in immunocompromised patients. It is likely to be missed or underreported due to lack of clinical awareness. MALDI-TOF MS is helpful in rapid identification. R. mannitolilytica is resistant to many routinely used antibiotics, including carbapenems.

There are currently no clear treatment guidelines or Clinical and Laboratory Standards Institute (CLSI) breakpoints for R. mannitolilytica. Treatment is challenging, as this species is frequently resistant to many antibiotics [1]. Resistance to many of the OPEN ACCESS β-lactam class of antibiotics, including the carbapenems, is generally observed in R. mannitolilytica. blaOXA-22 and blaOXA-60 are class D carabapenmase genes that are commonly associated with Ralstonia species [14]. Treatment is based on the antibiotic susceptibility profile of the isolate [1]. There are very few case reports on this rare opportunistic pathogen in bloodstream infections from India. In this case series, we summarize the clinical characteristics of 14 patients with nosocomial bloodstream infections caused by R.mannitolilytica in the last 2 years, and analyse their risk factors, management and outcomes. We have also reported drug susceptibility patterns, which may help in the management of patients infected with this bacterium.

CASE SERIES
This case series includes clinically significant non-duplicate culture isolates of Ralstonia species obtained from suspected bloodstream infections in microbiology laboratory of a tertiary care centre of northern India. These cases occurred between June 2019 and June 2021 in the Departments of Haematology, Critical Care medicine, Gastro-medicine, Pulmonary Medicine and Endocrine Surgery. Written informed consent for publication of clinical details was obtained from the patients before the study. A study proforma was designed, which included patient demographic data, clinical details, underlying risk factors during the episode of infection, duration of hospital stay, treatment received and outcomes.

Sample processing and identification of isolates
Blood samples were inoculated into aerobic and anaerobic blood culture bottles (Becton Dickinson Diagnostics, USA) and incubated in the in BACTEC blood culture system (BD Diagnostics, USA). Once flagged positive, a Gram stain was performed from the bottle and the broth was plated onto 5 % sheep blood agar and MacConkey agar. The plates were incubated at 37 °C in ambient air and then inspected for growth at 24 h and again at 48 h. All of the culture media used were obtained from HiMedia Laboratories (Mumbai, India). The bacterial isolates were first identified using the routine staining and biochemical tests used in our laboratory [15]. The biochemical reactions for this Gram-negative bacillus revealed the results as follows: catalase-positive, oxidase-positive, motile, non-fermenting, methyl red-negative, Voges-Proskauer-negative, indole-negative, triple sugar iron agar -K/K (alkaline/alkaline), and urease-negative. Citrate was utilized but aesculin and gelatin were not hydrolyzed. R. mannitolilytica and Ralstonia pickettii were differentiated on the basis of nitrate reduction (negative in R. mannitolilytica) and acidification of d-arabitol and mannitol (both negative in R. pickettii) [16]. The identity of all the isolates was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using the VITEK MS system (bioMérieux, Marcyl'Etoile, France). Briefly, one bacterial colony of each isolate was spotted directly onto a single well of a disposable, barcode-labelled Vitek MS-DS target slide (bioMérieux, Marcy l'Etoile, France) and overlaid with 1 µl of saturated α-cyano-4-hydroxycinnamic acid (CHCA) (Vitek MS-CHCA, bioMérieux, Marcy l'Etoile, France) matrix and then air-dried. Escherichia coli ATCC 8739 was used as the quality control strain and was transferred directly to designated spots on the target slide as per the recommendation of the manufacturer. The target slide with all prepared isolates was then loaded into the VITEK MS system to acquire the mass spectra of bacterial proteins [17]. Finally, the mass spectra acquired for each isolate were compared to the known mass spectra contained in the SARAMIS database. The software compares the spectra and generates a numerical value (score value) based on the similarities between the observed and stored data sets. A score value above 2.0 is generally considered to be a valid species level identification and values between 2.0 and 1.7 represent reliable genus-level identification.

Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed using the Kirby-Bauer disc diffusion method on Müller-Hinton agar and with an automated method (Vitek 2, bioMérieux) [18]. Antibiotic sensitivity was tested as per the Clinical and Laboratory Standards Institute (CLSI) guidelines [19]. As there were no CLSI breakpoints or zone diameters available for R. mannitolilytica, the results were interpreted using the CLSI breakpoints for Pseudomonas spp. [19]. P. aeruginosa ATCC 27853 and E. coli ATCC 25922 were put up as controls.

Environmental surveillance sampling
Environmental samples were collected from different wards from which R. mannitolilytica isolates had been obtained by an infection control nurse. Commercially available sterile swabs (HiMedia Laboratories, Mumbai, India) were used to collect samples from the patients' immediate surroundings, bed rails, tubing and medical devices. Samples from unused sterile intravenous fluids, liquid soaps and disinfectants and water (drinking water and tap water) were obtained in a sterile universal container. Air sampling was performed using a sieve impactor.

Processing of environmental samples
Swabs were incubated in brain heart infusion (BHI) media at 37 °C for 18-24 h. After 24 h, BHI media was inspected visually for any turbidity or growth. A small volume of sample was then taken with the help of an inoculating loop and sub-cultured on blood and MacConkey agar. The culture plates were further incubated for 24 h at 37 °C. Any positive growth was further identified using Gram staining and appropriate biochemical tests.

Statistical analysis
Statistical tests were performed using SPSS for Windows version 14 (SPSS, Inc., Chicago, IL, USA) for descriptive statistics. Categorical data were described using numbers and percentages.

RESULTS
A total of 14 cases of R. mannitolilytica were reported from our hospital over a period of 2 years. All of the patients presented with symptoms of fever and/or chills, and a positive blood culture for R. mannitolilytica. We carried out comprehensive environmental sampling from various sites in our hospital. However, after 48 h of incubation all of the cultures were found to be sterile. The age of the patient population ranged from 8 to 58 years with a median age of 15.5 years. The number of males and females enrolled in the study were 10 and 4, respectively, with a M : F ratio of 2.5 : 1. Detailed demographic and clinical characteristics of the patients are illustrated in Table 1. The majority of the patients with R. mannitolilytica infection had haematological malignancy (8/14) and there was one patient each with breast carcinoma, dengue haemorrhagic shock syndrome, decompensated chronic liver disease, acute respiratory distress syndrome, acute necrotizing pancreatitis and severe coronavirus disease 2019 (COVID- 19) pneumonia. The average duration of hospital stay was 29 days (range 10-58 days) and the mean time of development of infection after hospitalization was 15.1 days. Chemotherapy (9/14), mechanical ventilation (4/14) and steroid (2/14) use were the most common risk factors in these patients.
The following antibiotics were administered to patients after antibiotic susceptibility testing and according to the clinicians' decisions: cefoperazone/sulbactam was given to 10 (71.4 %) patients, meropenem to 3 (21.4 %) patients and ceftazidime to 1 (7.2 %) patient. Thirteen patients recovered with antibiotic therapy and one patient succumbed to his illness, but the cause of death for this patient was complications of COVID-19 (Table 2).

DISCUSSION AND CONCLUSION
Few cases of infections caused by R.mannitolilytica have been reported due to limited awareness of the pathogen. The development of modern medical care, inappropriate and unnecessary use of broad-spectrum antibiotics, and the extensive use of various immunosuppressants have caused increased rates of opportunistic infections from organisms such as R.mannitolilytica. The prevalence of Ralstonia infection is increasing notably, even without person-to-person transmission [1]. Ralstonia exist widely in external aqueous environments, including municipal water and medical water purification systems [20,21]. As the bacteria can pass through 0.2 µm filters during the sterilization process, medical products may be contaminated during the manufacturing phase [22]. Ralstonia can create biofilms on the surfaces of medical supplies and produce toxins [13]. Most infectious cases caused  by Ralstonia species are due to the use of contaminated solutions, chlorhexidine, saline solution, blood products and sterile water as well as the colonization of medical devices (tap water and water used for haemodialysis, bronchoscope flushing and heparin for flushing) [3,23,24].
In this case series we have described 14 nosocomial cases of bloodstream infections caused by R. mannitolilytica, although we could not track their source. All of our patients were at high risk of infection. The high-risk factors listed in the literature are cancer, blood vessel catheters, mechanical ventilation and other immunocompromised conditions [1,25] that were also found in our study. The most frequent risk factor in the study population was the use of chemotherapy and steroids, which could be why most of the patients in the study had malignancy, which itself is an immunocompromised state [3]. Further, these patients received chemotherapy as well as immunosuppressant drugs [3]. Hence, such patients are vulnerable to opportunistic infections such as Ralstonia, which is non-harmful to healthy persons [1]. Long-term use of steroids also causes an increase in the rate of infections due to deranged cellular immunity. Central lines are essential in malignancy patients for long-term chemotherapy infusions, and infection of central lines might happen during the insertion procedure as well as during the maintenance period. Biofilm formation in the central lines might cause bacteria to harbour and cause central line-associated bloodstream infections [4]. Blood products through central lines might help in the formation of biofilms that might cause central line infections in these cases. It is well accepted that, over time, the presence of endotracheal tubes for mechanical ventilation increases the risk of bacterial colonization and the development of infection. Similarly, it is conceivable that Ralstonia, a water-borne bacterium cultured from respiratory devices, may have the capacity to colonize the airways; its capacity to generate biofilms, like Pseudomonas species, enables it to cause infection in patients who are mechanically ventilated for longer periods [21]. Diabetes itself causes increased infections due to unbalanced blood sugar levels in patients. All of these patients had prolonged hospital stays, which might have led to acquisition of this bacterium. Boattini et al. (2018) reported that this pathogen is an important cause of nosocomial bacteraemia in diabetics, preterm infants, solid organ and haematological malignancy patients, and patients with end-stage renal disease [4]. Thomas et al. (2021) reported it to cause bacteraemia and gastroenteritis in a patient with rheumatoid arthritis.
Diagnosis and management of Ralstonia spp. infections is challenging. First, there is the difficulty of correctly identifying and differentiating between Ralstonia spp. members using routine laboratory analyses, because they have very similar biochemical patterns to each other and to other bacterial genera, such as the Burkholderia cepacia complex [1]. MALDI-TOF showed good performance regarding the identification of R. mannitolilytica in a previous study using isolates from patients with cystic fibrosis [26]. Although 16S rDNA is the reference method for identifying micro-organisms, it is costly and cumbersome. In the present study, MALDI-TOF correctly identified all of the isolates rapidly. Therefore, in comparison to the turnaround time and PCR identification cost of the 16S sequencing method, MALDI-TOF MS would be a better choice for identification of R. mannitolilytica . Our results support the general consensus that MALDI-TOF MS can provide rapid and accurate results [27]. Second, R. mannitolilytica are frequently resistant to numerous different types of antibiotics, including several beta-lactams and most of the aminoglycosides [1]. The organism may produce various enzymes that can hydrolyze antibiotics. These can confer resistance to a broad range of antibiotics, including benzylpenicillin, narrow-spectrum cephalosporins, ceftazidime, aztreonam and the carbapenems [26]. As currently there are no clear treatment guidelines for R. mannitolilytica and the data from various case reports reveal a notable heterogeneity in the percentage of antibiotic resistance amongst R. mannitolilytica isolates, in the course of treatment we advocate using antibiotic susceptibility testing to adjust the use of antimicrobial agents. Our antibiotic susceptibility pattern showed maximum resistance to aminoglycosides and no resistance to cefoperazone/sulbactum. Hence most of our patients were treated with cefoperazone/sulbactum or a with some other third-generation cephalosporins. Daxboeck et al. reported carbapenem resistance in 12 out of their 30 strains [28], which is consistent with our study, where we found carbapenem resistance in 6 out of 14 isolates. Thus meropenem was only used for seriously ill patients whose antibiograms showed sensitivity to meropenem. A 93 % (13/14) recovery rate in the study patients supports the view that early diagnosis and early initiation of appropriate antibiotics are required for good outcomes in these patients.
Even though R. mannitolilytica is not recognized as a major pathogen, clinicians and microbiologists should pay attention to the potential of this opportunistic bacterium, which is able to cause bloodstream infections, as it has certain notable characteristics, such as multidrug resistance, the ability to survive in water supplies and resistance to disinfection practices. Prompt diagnosis and subsequent administration of antibiotics in line with antimicrobial susceptibility testing results are needed to clear infections. MALDI-TOF MS is helpful for rapid identification and R. mannitolilytica is capable of being resistant to many routinely used antibiotics, including carbapenems.

Funding information
The authors received no specific grant from any funding agency.